On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the α3β4- and α4β2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases

Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The α3β4 nAChR and α4β2 nAChR subtypes were used to prepare the chromatographic columns and [3H] epibatidine dihydrochloride ([3H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (Kd > 4700 nM for α3β4). The displacement of [3H] EB by each of the test compounds and internal controls was calculated and expressed as Δml. Functional studies were then conducted using a stably transfected cell line that expresses the α3β4 nAChR and EC50 values were determined for the test compounds and the internal controls. A comparison of the Δml and EC50 values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the α3β4 nAChR. A similar comparison could not be made for the α4β2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.